Several findings in this reporting period relate to potential treatments to slow or prevent catecholaminergic neurodegeneration. We are especially interested in combining N-acetylcysteine (NAC) with a monoamine oxidase inhibitor (MAOI), to test the catecholaldehyde hypothesis, dietary manipulations that might be beneficial, and treatments of neurogenic orthostatic hypotension (nOH), which is a common consequence of catecholaminergic neurodegeneration in Lewy body diseases. (1) N-Acetylcysteine (NAC) mitigates DOPAL-induced protein modifications: 3,4-Dihydroxyphenylacetaldehyde (DOPAL), an obligate intermediary metabolite of dopamine, The is an autotoxin that may contribute to catecholaminergic neurodegeneration. Mechanisms of DOPAL toxicity may include misfolding of intracellular proteins such as alpha-synuclein (AS). We found that DOPAL potently oligomerizes AS, converting AS to a toxic form, and also forms quinone-protein adducts with (quinonizes) AS. Co-incubation with NAC mitigated or prevented these alterations. These data help rationalize treatment with NAC and a monoamine oxidase inhibitor (MAOI). The MAOI would inhibit DOPAL formation by decreasing enzymatic oxidation of dopamine, and NAC would inhibit formation of harmful quinones by decreasing spontaneous oxidation of DOPAL to DOPAL-quinone (Jinsmaa et al., JPET 2018;366:113-124). In a follow-up study we have found preliminarily that near-infrared fluorescence microscopy visualizes intra-cellular quinoproteins evoked by DOPAC in cultured human oligodendrocytes and that NAC decreases this effect (Jinsmaa et al., unpublished observations). (2) Poor plasma bioavailability of DOPET after olives ingestion: Olives contain abundant 3,4-dihydroxyphenylethanol (hydroxytyrosol, DOPET), a potential nutraceutical. After healthy volunteers ate 10 olives, plasma DOPET increased; however, there were much larger increases in plasma 3,4-dihydroxyphenylacetic acid (DOPAC), a liver metabolite of DOPET. The plasma bioavailability of ingested DOPET seems to be poor due to a hepatic first pass effect (Goldstein et al., Clin Transl Sci 2018;11:32-37). (3) Substantial renal conversion of L-threo-3,4-dihydroxyphenylserine (L-DOPS) to norepinephrine in patients with neurogenic orthostatic hypotension: The norepinephrine precursor L-DOPS is approved for treatment of symptomatic orthostatic hypotension (OH). L-DOPS-induced increases in plasma norepinephrine levels are too small to explain the pressor effect of the drug. The kidneys contain abundant L-aromatic-amino-acid decarboxylase, which catalyzes the conversion of L-DOPS to norepinephrine. We assessed renal norepinephrine production from L-DOPS by comparing norepinephrine/L-DOPS ratios in urine to those in plasma in patients on L-DOPS. The ratio of norepinephrine/L-DOPS in urine averaged 63 times that in plasma. Thus, there is extensive renal production of norepinephrine from L-DOPS (Lamotte et al., Clin Auton Res 2019;29:113-117). Collaborations: (a) MAOI+NAC treatment in the rat rotenone animal model of PD: We discovered and reported previously that in synucleinopathies DOPAL buildup in the putamen reflects a double hit of decreased vesicular uptake of cytoplasmic dopamine and decreased enzymatic detoxification of DOPAL by aldehyde dehydrogenase (ALDH). In collaboration with Yehonatan Sharabi (Tel Aviv University) we are testing the catecholaldehyde hypothesis in animals by assessing whether MAOI+NAC treatment prevents catecholaminergic neurodegeneration in the rotenone PD model. (b) Mesenchymal stem cells to treat multiple system atrophy: Multiple system atrophy (MSA) is a rare neurodegenerative disease characterized by chronic autonomic failure and AS deposition in glial cytoplasmic inclusions in the brain. Intrathecal injection of mesenchymal stem cells might exert beneficial neurotrophic effects in this otherwise invariably lethal disease. In a collaborative study with investigators at the Mayo Clinic we are tracking cerebrospinal fluid catechols as a neurochemical biomarker of the treatment. (c) Renal sympathetic ablation to treat refractory hypertension: Renal sympathetic ablation is a possible non-pharmacologic treatment for refractory hypertension. In a collaborative study with Benjamin Levine (Univ. of Texas) under NIH Clinical Protocol 03-N-0004 we used 18F-dopamine and 11C-methylreboxetine scanning to assess the extent of local denervation in a clinical trial of renal sympathetic ablation to treat refractory essential hypertension. This pilot study yielded negative results. (d) Effects of atomoxetine on cerebrospinal fluid and plasma catechols: Atomoxetine is an FDA-approved drug that inhibits the cell membrane norepinephrine transporter (NET). Dopamine is a better substrate for the NET than is norepinephrine. As part of a collaborative study with Allan I. Levey (Emory) about whether atomoxetine improves cognitive dysfunction in people with mild cognitive impairment, we assayed cerebrospinal fluid (CSF) and plasma levels of catechols. Preliminarily, in both CSF and plasma atomoxetine treatment highly significantly increased levels of dopamine and norepinephrine without increasing levels of their respective neuronal metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenyglycol (DHPG). Increases in CSF and plasma dopamine were particularly striking. These findings raise the possibility that atomoxetine might increase dopamine delivery to its receptors in the nigrostriatal system and thereby alleviate motor manifestations of PD. (e) Adeno-associated virus (AAV)-mediated gene therapy in an animal model of Menkes disease: Menkes disease is a rare X-linked recessive disorder of a copper ATPase gene (ATP7A). Since dopamine-beta-hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine, is a copper enzyme, increased levels of dopamine and its metabolites with respect to norepinephrine and its metabolites provides diagnostic biomarkers of Menkes disease in at-risk newborns. In a collaborative study of a mouse model of Menkes disease, we found that AAV9-mediated gene therapy in the form of cerebrospinal fluid-directed rAAV9-rsATP7A plus subcutaneous copper histidinate improved survival and outcomes (Haddad et al., Mol Ther Methods Clin Dev 2018;10:165-178). (f) AAV-mediated gene therapy in an animal model of PD: Dopamine deficiency causes the movement disorder that defines PD. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis, and GTP cyclohydrolase-1 (GCH-1) is a key enzyme in the production of tetrahydrobiopterin, a required co-factor for TH. We carried out assays of CSF and tissue catechols in an exploratory study of intrathecal AAV2-TH/GCH1 in a rat PD model under a Materials Cooperative Research and Development Agreement. (g) ALDH inhibition to treat opiate abuse: Opiate abuse is a major, increasing public health burden. Administration of opiates increases dopamine release in a particular brain system involved with reinforcement and learning. A possible treatment is to reduce rewarding effects of opiates by inhibiting dopamine synthesis. According to the hypothesis that ALDH inhibition leads to production of tetrahydropapaveroline, which feedback-inhibits TH, under the Helping to End Addiction Long-term (HEAL) initiative a clinical trial of an ALDH inhibitor is under way at the NIH Clinical Center. To determine whether the drug actually inhibits ALDH, we are collaborating by assaying plasma samples of protocol participants before and after drug administration. Preliminarily, we did not obtain neurochemical evidence for ALDH inhibition, with the dosing regimen being used.